1. Field of Invention
This invention relates to one-step methods for conducting assays of fluids suspected of containing selected ligands. It further relates to an apparatus for conducting these assays.
2. Description of the Prior Art
In recent years, immunoassay procedures have undergone nearly continuous refinement in an effort to simplify and speed the process of detecting selected ligands in fluids suspected of containing them (these fluids will hereafter be referred to as analyte samples). As a result of this work, assays using monoclonal antibodies which recognize different epitopic sites on an antigen have been developed which made it possible to conduct simultaneous assays without repeated incubation and washing of bound antibody.
One example of such a process can be found in U.S. Pat. No. 4,632,901. In the method of that patent, a liquid sample suspected of containing an antigen of interest is poured onto a porous material to which an antibody capable of binding the antigen of interest is coupled. Through capillary action of an absorbent material in contact with the porous material, the sample is drawn in a directed fashion through the porous material and past the coupled antibody. Antigen in the sample will be captured by, and will bind with, the coupled antibody.
After this first step is completed, a second step is taken wherein a separate solution of labelled antibody is passed through the porous material so as to bind to the antigens which are now bound to coupled antibody. Unbound labelled antibody is then flushed from the porous member by a washing step, which may be followed by an incubation period. Finally, a third solution containing substrate reactive with the label on the antibody of the second solution is added to cause a visible color change indicative of the presence of the antigen of interest. To facilitate accurate performance of this method, an apparatus is provided which funnels sample through to the absorbent member which, by capillary action, will pull the sample through the device and, in one embodiment, out of its bottom.
Similar to the three step method of U.S. Pat. No. 4,632,901 is the dipstick method of U.S. Pat. No. 4,366,241. In the dipstick method, the assay device is a strip on which an immunological ligand is bound. The strip is dipped into or otherwise contacted with the analyte sample, then successively contacted one or more separate solutions which may contain a labelled antiligand, enzyme substrate (for enzyme labels) and/or washing fluids to remove any unbound label. A filtration step may also be taken to facilitate complete washing (see, e.g., U.S. Pat. No. 4,623,461). Other researchers have suggested the use of a second control assay for verification of the assay results (see, e.g., U.S. Pat. No. 4,200,690 [method provided for analyzing fecal samples using two washing and incubating steps]).
For a similar dipstick device with both labelled and unlabeled antibody bound to the sample-receiving porous material (see PCT Application No. PCT/GB88/0322).
While the developments described above have provided compact, relatively efficient means for performing assays, several problems with the presently known procedures remain. First, after addition of the analyte sample to the assay device, these procedures require the addition or use of one or more additional solutions to complete the assay. As each solution is added to the device, or each device dipped into successive solutions, the opportunities for spillage or contact between the solution(s) and the user are enhanced. This problem is further worsened in assays which require the user to remove a portion of the device (such as a viewing port) during the procedure.
Multiple dipping or pouring steps present not only a possible loss of sensitivity of the assay (through loss of reagent solutions), but also an aesthetic and hygienic problem for the analyst. Consumers in particular are likely to be intimidated by the need to mix and add one or more solutions during the procedure, if not put-off altogether by the prospect of contacting the analyte sample (which is usually urine in consumer assays such as pregnancy tests).
Another consideration is how accurately the results of the assay are reported. Chromatographic devices are becoming more common in recent years. For example, U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537 describe test strip devices where different zones in the strip contain reagents needed to produce a detectable signal as analyte passes into or through the zones.
A variation on the strip devices described above requires the user to apply a second ligand containing fluid to the device after application of the analyte sample. A fluorescent or enzymatic label is covalently bound to the ligand in the second fluid; the ligand is specific to the analyte of interest and will bind thereto, allowing its detection on activation of the bound label. This and similar devices (see, e.g., U.S. Pat. No. 4,740,468) suffer to some extent from the same shortcoming as the devices which require washing and incubation steps; the need to use more than one solution at the correct time and in the correct order to perform the assay.
One effort to avoid the need for addition of reagent solutions found in prior art devices is reflected in U.S. Pat. No. 5,075,078. This patent describes several embodiments of chromatographic test strip devices which use a visually detectible result format generating plus (+) or minus (-) signs to indicate, respectively, the presence or absence of the analyte of interest in the sample.
In particular, the '078 devices are directed to solving the problem of "leading edge effects" i.e., where the plus or minus sign is not clearly or completely formed, causing the test results to be inconsistent or unreadable. To explain, conventional plus/minus assay devices include a chromatographic substrate of absorbent material. A test region will be located on the substrate, in which is located a procedural control bar parallel to the direction of fluid flow through the substrate and a patient test bar perpendicular to the control bar. Typically, a ligand specific to the analyte of interest will be bound within the test bar which will capture analyte present in the sample fluid. A label reagent is added to the device after addition of the analyte sample fluid; alternatively, the label reagent may be added with the sample fluid.
If the analyte of interest is present in the sample fluid, the label reagent will react with it in the test bar and with a corresponding ligand in the control bar, forming a plus sign. In absence of the analyte of interest, the label reagent will react only with the ligand bound in the control bar, forming a minus sign. However, experience with these prior art devices has shown that several failures can occur which produce misleading test results. For example, depletion of ligand in the control bar can prevent binding of label in that area, causing only part of the minus sign to appear. Another failure is caused by a solvent front which causes the edges of the control and/or test bars to become blurred; it is this phenomenon which is commonly referred to as a leading edge effect.
The plus/minus devices of the '078 patent attempt to avoid these failures by placing the control and test bars at a preferred 45.degree. angle to the direction of fluid flow and at a 90.degree. angle with respect to each other. A diffusible label application pad is included in the device which includes a label conjugated to a ligand (generally, an antibody). The device is described as being "self-performing" (i.e., requiring only the addition of analyte sample fluid to perform the test) and as producing clear test results (due to the orientation of the test and control bars, which is intended to minimize the distance the sample fluid must travel, thus limiting the opportunity for leading edge effects to affect the test results).
However, whether or not leading edge effects are controlled by the '078 patent's orientation of the control and test bars, the possibility for misleading results is still inherent in the device. Specifically, placement of the displays for positive and negative test results on the same region of the substrate by definition allows for the two results to be confused. Accordingly, a test device which provides for separate test result displays which cannot be confused is still needed in the art. Further, the device must still be user-friendly; i.e., require a minimum of steps to perform and allow those steps to be performed in a simple, hygienic manner. These needs are addressed by the present invention.